Recurrence of breast cancer following treatment of a primary tumor is the principal cause of mortality in breast cancer patients. However, little is known about the biological mechanisms underlying recurrence, in large part due to the dearth of in vivo models of human breast cancer dormancy and spontaneous recurrence. To address this, our laboratory has established doxycycline-inducible bitransgenic mouse models that develop invasive mammary carcinoma following doxycycline administration, including the MMTV-rtTA~TetO-neu (MTB/TAN) bitransgenic mouse. Upon oncogene down-regulation induced by doxycycline withdrawal, mammary tumors regress to a non-palpable state, but leave foci of dormant residual neoplastic cells that ultimately give rise to spontaneous recurrent tumors~ these dormant foci are termed minimal residual lesions (MRLs). Notably, MRLs contain dormant tumor cells juxtaposed with tumor-associated macrophages (TAMs). This finding is particularly intriguing given recent clinical data indicating that macrophages are recruited to breast cancers following treatment, and that macrophage number is an independent negative prognostic factor for recurrence-free survival in breast cancer patients. While TAMs have been suggested to promote primary breast cancer progression through a variety of mechanisms, a functional role for macrophages in tumor dormancy or recurrence has yet to be identified. We propose a model in which TAMs in MRLs establish a collagen-rich and immunosuppressive niche that supports dormant tumor cell survival and recurrence. This model predicts that alteration of this TAM phenotype would inhibit recurrence by disrupting the microenvironment of dormant tumor cells. As macrophage phenotype is plastic, alteration of TAM phenotype is an attractive therapeutic target. In this regard, recent research suggests that CD40 agonist therapy can induce an M1 state in TAMs, and that these macrophages can subsequently inhibit tumor growth. Additionally, we predict that dormant tumor cells and macrophages co- migrate along collagen fibrils within the MRL, which may explain the co-occurrence of dormant disseminated tumor cells with circulating tumor cells in breast cancer patients. The central hypothesis of this proposal is that TAMs are required for tumor dormancy and recurrence by means of their ability to modulate the MRL microenvironment. As such, we hypothesize that activation of the CD40 pathway in MRL TAMs will induce an M1 phenotype, thereby disrupting the dormant tumor cell microenvironment and inhibiting recurrence. Specifically, we propose that administration of the anti-CD40 agonist antibody, FGK45, following oncogene down-regulation will generate an M1 phenotype in TAMs, thereby altering the collagen-I content and immune milieu of the MRL in a manner that promotes tumor cell death and prevents tumor recurrence. Together, we believe these studies will identify a functional role for macrophages in minimal residual disease and mammary tumor recurrence.